It is desirable to be able to separate proteins in biological samples, as well as to subsequently characterize the separated proteins. In a first example, the presence and quantity of particular proteins (i.e., marker proteins) can be used to detect and determine the extent or severity of a disease or other abnormality in an individual. In a second example, the blood of an animal (i.e., a wild type or a transgenic animal) may contain a quantity of a protein of interest (e.g., the product of a transgene). In such a case, it would be convenient to obtain blood from the animal and separate the desired protein or proteins from the other blood proteins. In general, protein separation and characterization can be carried out using any convenient method(s), such as, for example, electrophoresis (e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis or SDS-PAGE) and subsequent immunoblotting (e.g., Western blotting).
However, separation/characterization of proteins in serum and plasma is hampered by the presence at high levels of such proteins as albumin and immunoglobulin, which non-specifically bind (“stick”) to other proteins. It is known in the art that, when serum is loaded and run on an SDS gel, albumin (blood's primary carrier protein) causes many artifacts, such as smearing, a collapsed lane appearance, and the like (see FIG. 1). The effectiveness of analysis of serum is also limited because such serum proteins often bind to marker proteins, and hence interfere with the migration of the marker proteins on a gel. For an effective analysis, therefore, a marker protein must be separated from any other serum protein that may interfere with its migration on the gel. In enzyme linked immunosorbent assays (ELISAs) and other assays where denaturing is not performed, binding of antibody to a target epitope on the marker protein can be inhibited by the target epitope being hidden by the binding of other proteins to the marker protein. In cases with low serum level of marker protein, such as mild or chronic disease states, where the amount of target epitope is low, the binding by other serum proteins may reduce access to target epitope enough to cause false negative results.
For example, the myofilament proteins cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are biochemical cardiac markers frequently used in the assessment of acute coronary syndrome (ACS) and other myocardial injuries. cTnI and cTnT are not present in the blood of normal, healthy individuals. However, in addition to ACS, elevated marker levels have been found in the blood in cases of congestive heart failure (Missov et al. Circ. 1997 96:2953-2958; Missov and Mair Am. Heart J. 1999 138:95-99), unstable angina (Ottani et al. Am. Heart J. 1999 137:284-291), pulmonary embolism (Giannitsis et al. Circ. 2000102:211-217), myocarditis (Lauer et al. J. Am. Coll. Cardiol. 1997 30:1354-1359), sepsis and septic shock (ver Elst et al. Clin. Chem. 2000 46: 650-657), as well as in patients undergoing percutaneous intervention (Tardiff et al. J. Am. Coll. Cardiol. 1999 33:88-96), cardiac surgery (McDonough et al. Circ. 2001 103:58-64) or implantable cardioverter defibrillator shock application (Schluter et al. Clin. Chem. 2001 47:459-463). cTnI and cTnT in serum have been reported to represent myocardial damage and increased risk of future adverse outcomes (Jaffe et al. Circulation 2000 102:1216-1220). However, problems exist with many of the commercially available detection kits currently in use for these biomarkers.
For example these kits may or may not detect all of the cTnI depending upon the combination of antibodies provided. Further, commercial kits can not differentiate whether or not modified forms of cTnI are present. Nor can these kits identify which forms of cTnI are in the sample. Another problem is that proteins in serum are bound to each other as well as to other proteins which can mask or hide epitopes, thus rendering the proteins undetectable by the antibodies of the kit. Thus, for some patients experiencing an acute myocardial infarction (AMI), the current cTnI kits do not detect all of the intact cTnI and modification products thereof that are released into the serum.
In addition, current cTnI kits are not always capable of detecting intact cTnI and modification products thereof in cardiac patients who are not experiencing an AMI, as the commercially available assays appear to have low analytical and diagnostic sensitivity.
These patients can be discharged from emergency with a diagnosis of chest pain/not yet diagnosed.
Accordingly, the ability to separate a protein of interest from other proteins in biological samples is important to the development of diagnostic kits with higher analytical and diagnostic sensitivity.
Separation of the protein of interest from other proteins like albumin is also desirable in the case of protein purification from blood, serum or plasma.
Unfortunately, known methods for removing native serum proteins from serum/plasma under native conditions (e.g., non-denaturing, non-reducing conditions, such as immunoprecipitation, acid extraction, gel filtration, ion exchange chromatography) typically lead to substantial loss of the marker protein or proteins, due in large part to the above-mentioned non-specific sticking. In methods where a marker protein or proteins is completely purified from native serum/plasma, analysis of a marker protein level or levels relative to level of other proteins is rendered impossible.